1. Field of the Invention
The invention relates to pharmaceutical compositions containing TSG-6 protein, inducible in connective tissue cells by tumor necrosis factor or interleukin-1, and methods for treating inflammatory diseases and disorders, or cancer-related pathologies.
2. Description of the Background Art
Tumor necrosis factor (TNF) is a powerful pleiotropic cytokine important in host defenses against tumors and infectious agents. TNF has also been implicated in the pathology of some neoplastic diseases, infections and autoimmune disorders as well as in various pro-inflammatory actions which result in tissue injury. TNF is an extremely "versatile" and clinically significant cytokine. Most of its actions are likely to be mediated by the activation or inactivation of specific genes in the cells upon which it acts. One exception to this mode of action is the rapid cytotoxic effect of TNF on certain target cells; this effect is augmented by inhibitors of RNA or protein synthesis and does not appear to depend on the modulation of gene expression (Matthews, N., Br. J. Cancer 48:405 (1983)). Many specific gene products have been shown to be up-regulated in TNF-treated cells.
A TNF-stimulated gene, abbreviated as TSG-6 gene, was originally isolated from a cDNA library prepared from TNF-treated normal human fibroblasts (Lee et al., Mol. Cell. Biol. 10:1982-1988, 1990). Early studies showed that TSG-6 gene transcription was induced by the pro-inflammatory cytokines TNF and IL-1 (Lee et al., J. Cell Biol. 116:545-547, 1992) or by bacterial LPS (Wisniewski et al., J. Immunol. 151:6593-6601, 1993) in fibroblasts, mononuclear cells, chondrocytes and synovial cells. TSG-6 codes for a secretory 35-kDa glycoprotein, designated TSG-6 protein, that is a member of the hyaladherin family of hyaluronan binding proteins (Lee et al., supra, 1992; Toole, In Cell Biology of Extracellular Matrix, Hay (ed.), 2nd ed., Plenum Press, New York, p. 305-341, 1991). Sequencing of the cDNA revealed an open reading frame coding for a polypeptide of 277 amino acids including a cleavable signal peptide (Lee et al., supra, 1992). In its N-terminal half, the predicted amino acid sequence shows 36-40% homology to members of the hyaladherin family of proteins that includes the lymphocyte homing/hyaluronan receptor CD44, cartilage link protein, and the proteoglycan core proteins aggrecan and versican (Lee et al., supra, 1992). The C-terminal half of TSG-6 shares 30% sequence homology with the A chain of complement Clr. This homology region forms a so-called CUB domain which is a motif found in proteins that are developmentally regulated (Bork et al., J. Mol. Biol., 231:539-545, 1993). The rabbit homologue of TSG-6 was recently cloned and shown to be a developmentally regulated protein exhibiting 94% identity to human TSG-6 at the amino acid level (Feng et al., J. Biol. Chem., 268:9387-9392, 1993).
Two N-glycosylation consensus sequences are present in TSG-6, and the presence of N-linked carbohydrate was experimentally confirmed (Lee et al., supra, 1992). Like other hyaladherins, TSG-6 protein binds specifically to hyaluronan. TSG-6 expression is tightly regulated, with its transcription in fibroblasts rapidly activated by stimulation with the pro-inflammatory cytokines IL-1 or TNF-.alpha. (Lee et al., supra, 1990; J. Biol. Chem. 268:6154-6160, 1993). High levels of TSG-6 protein were found in synovial fluids of patients with rheumatoid arthritis and some other forms of arthritis, whereas no TSG-6 protein was detected in synovial fluids from normal human joints (Wisniewski et al., supra, 1993). In addition, synoviocytes from the joints of rheumatoid arthritis patients showed constitutive TSG-6 expression that was further upregulated by IL-1 and TNF, cytokines that are regularly found in the rheumatoid synovial fluid or tissue (Wisniewski et al., supra, 1993).
TSG-6 binds firmly and selectively to a discrete protein present in animal sera (Wisniewski et al., In Physiology and Pathophysiology of Cytokines, Mantovani et al. (eds.), Biomedical Press, Augusta, Ga., p. 149-155, 1992; supra, 1993; Biochemistry 33:7423-7429, 1994). The complex of TSG-6 with its binding protein shows a molecular size of .about.120 kDa on SDS-PAGE; the presence of this complex was also demonstrated in synovial fluids of arthritis patients (Wisniewski et al., supra, 1993). The TSG-6 binding protein was purified from human serum and shown by microsequencing to be identical to inter-.alpha.-inhibitor (I.alpha.I) (Wisniewski et al., supra, 1994).
Inter-.alpha.-inhibitor (I.alpha.I) is a member of a family of closely related proteins with serine proteinase inhibitory activity (Gebhard et al., Biol. Chem. Hoppe-Seyler, 371: Suppl. 13-22, 1990; Pratt et al., Biochemistry 26:2855-2863, 1987), consisting of I.alpha.I, pre-.alpha.-inhibitor (P.alpha.I), and inter-.alpha.-like inhibitor (I.alpha.LI) (Enghild et al., J. Biol. Chem. 264:15975-15981, 1989; Gebhard et al., supra, 1990; Rouet et al., Biol. Chem. Hoppe-Seyler 373:1019-1024, 1992). The protease inhibitory activity of these proteins resides exclusively in a polypeptide chain termed bikunin (Gebhard et al., Eur. J. Biochem. 181:571-576, 1989; Gebhard et al., supra, 1990) that is shared by all members of this family. The different proteins are formed by the linkage of bikunin to one or two of three heavy chains (HC1, HC2, HC3) which show 38-54% amino acid sequence homology to each other (Bourguignon et al., Eur. J. Biochem. 212:771-776, 1993). I.alpha.I consists of bikunin linked to both HC1 and HC2, P.alpha.I consists of bikunin linked to HC3, and I.alpha.I is a complex of bikunin with HC2 (Enghild et al., supra, 1989; Gebhard et al., supra, 1990; Rouet et al., supra, 1992). A chondroitin 4-sulfate chain whose reducing end is linked to Ser.sup.10 of bikunin (Enghild et al., J. Biol. Chem. 266:747-751, 1991; Chirat et al., Int. J. Biochem. 23:1201-1203, 1991) cross-links the polypeptide chains of these complex protein molecules, which are all stable in SDS-PAGE under reducing conditions.
The bikunin chain is solely responsible for the protease inhibitory activity of I.alpha.I and of other members of this family. The protease inhibitory activity of I.alpha.I is specific for trypsin, chymotrypsin, plasmin, cathepsin G, acrosin, and leukocyte elastase (Steinbuch, Meth. Enzymol. 45:760-762, 1976; Jochum et al., Hoppe Seylers Zeitschrift fur Physiologische Chemie 364:1709-1715, 1983; Balduyck et al., Biol. Chem. Hoppe-Seyler 366:9-14, 1985; Bromke et al., Biochem. Med. 27:56-57, 1982, Lambin et al., Thrombosis Res. 13:563-568, 1978).
Although the trypsin-inhibitory activity of I.alpha.I has been known for a long time (Heide et al., Clin. Chem. Acta 11:82-85 (1965)), little is known about the functions of the different members of the I.alpha.I family. However, disease-associated presence in various tissues and fluctuations seen in the serum levels of I.alpha.I and I.alpha.I-related proteins suggest an involvement in pathologic processes. Daveau et al., Biochem. J. 292:485-4924 (1993) reported a distinct pattern of changes in serum concentrations of the different members of the I.alpha.I family during acute inflammation. Proteins identical with, or closely related to, the bikunin chain of I.alpha.I have been detected in stroma and the surrounding connective tissue of malignant tumors (Yoshida et al., Cancer 64:860-869 (1989)), in brain tissue of patients with Alzheimer's disease (Yoshida et al., Biochem. Biophys. Res. Commun. 174:1015-1021 (1991)), and in serum and urine of patients with inflammatory disease, cancer, and leukemias (Rudman et al., Cancer Res. 36:1837-1846 (1976); Franck et al., Scand. J. Clin. Lab. Invest. 43:151-155 (1976); Chawla et al., J. Cell Biochem. 42:207-217 (1990)). A link between I.alpha.I and rheumatoid arthritis was suggested over 20 years ago when Becker et al., Arthritis Rheum. (1971), found I.alpha.I associated with hyaluronan in the synovial fluid of patients with rheumatoid arthritis, whereas no I.alpha.I was detectable in control synovial fluids. This finding was confirmed and extended to show that I.alpha.I associates in vitro with hyaluronan isolated from the synovial fluid of healthy subjects (Hutadilok et al., Ann. Rheum. Dis. 47:377-385 (1988)). Huang et al., J. Biol. Chem. 268:26725-26730 (1993) showed recently that in the presence of serum the two heavy chains of I.alpha.I become covalently associated with hyaluronan.
In view of the inducibility of TSG-6 by the pro-inflammatory cytokines TNF and IL-1, as well as by LPS, it appeared likely that TSG-6 would play a role in inflammation. This view was reinforced by the demonstration that TSG-6 protein is present in the synovial fluids (and, at lower concentrations, also in the sera) of patients with rheumatoid arthritis and some other forms of arthritis (Wisniewski et al., supra, 1993). TNF, IL-1, and LPS are known to stimulate the synthesis of many other proinflammatory secreted proteins, including other cytokines, such as IL-8 (Matsushima et al., J. Exp. Med. 167:1883-1893, 1988), and the metalloproteinases collagenase and stromelysin (Brenner et al., Nature 337:661-663, 1989; Dayer et al., J. Exp. Med. 162:2163-2168, 1985; Quinones et al., J. Biol. Chem. 264:8339-8344, 1989). Therefore, the initial expectation was that TSG-6 protein would also turn out to have a pro-inflammatory function and that methods of treating inflammatory conditions would involve inhibiting the pro-inflammatory effect of TSG-6.
U.S. Pat. No. 5,386,013 discloses that the TSG-6 protein, encoded by a TNF-stimulated gene, is expected to have a pro-inflammatory effect as antibodies specific for TSG-6 protein would be used in a method of treating inflammatory conditions by binding to TSG-6 and inhibiting its activity.
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